Methods and compositions for use in the treatment of filovirus mediated disease conditions

ABSTRACT

Methods and compositions are provided for at least slowing the progression of a filovirus mediated disease condition in a host. In the subject methods, an effective amount of an agent that at least reduces the amount of folate receptor mediated filovirus cell entry is administered to the host. The subject methods find use in both the prevention and treatment of filovirus associated disease conditions, including Marburg and Ebola-Zaire virus mediated disease conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) toprovisional patent applications Serial Nos. 60/170,004, filed Dec. 9,1999 and 60/237,421, filed Oct. 2, 2000, each of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The field of this invention is virology, and in particularfiloviruses.

BACKGROUND OF THE INVENTION

[0003] Filoviruses belong to a virus family called Filoviridae and cancause severe hemorrhagic fever in humans and non-human primates. So far,only two members of this virus family have been identified: Marburgvirus and Ebola virus. Four subtypes of Ebola virus have beenidentified: Ivory Coast, Sudan, Zaire, and Reston. The Reston subtype isthe only known filovirus that does not cause severe disease in humans;however, it can be fatal in monkeys.

[0004] Filoviruses, including the Marburg and Ebola viruses, causesporadic epidemics of human disease characterized by systemichemorrhage, multi-organ failure and death in most instances. In anoutbreak or isolated case among humans, just how the virus istransmitted from the natural reservoir to a human is unknown. Once ahuman is infected, however, person-to-person transmission is the meansby which further infections occur. Specifically, transmission involvesclose personal contact between an infected individual or their bodyfluids, and another person. During recorded outbreaks of hemorrhagicfever caused by filovirus infection, persons who cared for or workedvery closely with infected individuals were especially at risk ofbecoming infected themselves. Nosocomial transmission through contactwith infected body fluids, e.g., via re-use of unsterilized syringes,needles, or other medical equipment contaminated with these fluids—hasalso been an important factor in the spread of disease. When closecontact between uninfected and infected persons is minimized, the numberof new filovirus infections in humans usually declines. Although in thelaboratory the viruses display some capability of infection throughsmall-particle aerosols, airborne spread among humans has not beenclearly demonstrated.

[0005] The onset of illness is abrupt, and initial symptoms resemblethose of an influenza-like syndrome. Fever, headache, general malaise,myalgia, joint pain, and sore throat are commonly followed by diarrheaand abdominal pain. A transient morbilliform skin rash, whichsubsequently desquamates, often appears at the end of the first week ofillness. Other physical findings include pharyngitis, which isfrequently exudative, and occasionally conjunctivitis, jaundice, andedema. After the third day of illness, hemorrhagic manifestations arecommon and include petechiae as well as frank bleeding, which can arisefrom any part of the gastrointestinal tract and from multiple othersites.

[0006] There is currently no accepted vaccine or direct therapy for theclinical manifestations of infection, other than general supportivemeasures. Interferon and ribavirin show no in vitro effect against theseagents. The case-fatality rate has been estimated to range from 30% to80%.

[0007] In view of the foregoing discussion, there is a need for thedevelopment of vaccine and/or treatment protocols for these types ofdisease conditions. The present invention addresses this need.

[0008] Representative Literature

[0009] Xu et al. (1998) Nat. Med. 4:37-42.

SUMMARY OF THE INVENTION

[0010] Methods and compositions are provided for at least slowing theprogression of a filovirus mediated disease condition in a host. In thesubject methods, an effective amount of an agent that at least reducesthe amount of folate receptor mediated filovirus cell entry isadministered to the host. The subject methods find use in both theprevention and treatment of filovirus associated disease conditions,including Marburg and Ebola-Zaire virus mediated disease conditions.

[0011] The invention further provides agents useful in treating afilovirus-mediated disease condition, as well as compositions comprisingthe agents. Agents include those that inhibit filovirus binding to afolate receptor on the cell surface, agents that reduce the level offolate receptor on the cell surface, and agents that modulate the folatereceptor such that binding of a filovirus to the folate receptor isreduced.

[0012] The invention further provides screening methods for identifyingagents that reduce filovirus entry into a susceptible cell. Bothcell-free and cell-based assay methods are provided.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIGS. 1A to 1C provide graphical results showing thatco-expression of an envelope negative HIV clone with a gene encodingMarburg or Ebola Zaire envelope glycoproteins leads to the generation ofinfectious virion particles with properties of the parental filovirus.

[0014]FIGS. 2A and 2B provide a graphical representation of the resultsof a permissivity screening assay.

[0015]FIG. 3 provides a graphical representation of the efficiency ofinfection of Jurkat EctR with a retroviral cDNA library.

[0016]FIG. 4 provides a table of the results of viability assay in whichJurkat EctR cells were challenged with a retroviral cDNA library and HIVblasti-pseudotype viruses after selection in 40 μg/ml blasticidin S.

[0017]FIG. 5 provides graphical results from an assay in which JurkatEctR batches selected for blasticidin S were rechallenged with VSV,Ebola-Zaire and Marburg luciferase pseudotype viruses.

[0018]FIG. 6 provides the graphical results of an assay in which theinfection of Jurkat EctR cells transiently expressing truncated humanfolate receptor alpha with a Marburg pseudotype virus was studied.

[0019]FIG. 7 provides a graphical representation of an assay in whichthe infection of target cells by Marburg and Ebola-Zaire pseudotypeviruses after phospholipase C treatment was studied.

[0020]FIG. 8 provides the graphical results of an assay in whichinfection of a Jurkat EctR clone derived from the initial blasticidinselection following infection with Marburg psedutotype virus wascompletely inhibited by a commercially available rabbit polyclonalantiserum (from Biogenesis) raised against the human folate bindingprotein.

[0021]FIG. 9 provides the graphical results of an assay in whichinfection of Vero E6 cells by MBG lucerfase virus, but not by VSVluciferase virus, is inhibited in the presence of polyclonal rabbitanti-bovine folate binding protein, but not by normal rabbit serum.

[0022]FIG. 10 provides the graphical results of an assay, showing thatentry by MBG, but not VSV, luciferase virus into Jurkat-EctR F10 cellsis specifically abrogated in the presence of mouse monoclonal anti-FR,compared to isotype control, antibody.

[0023]FIG. 11 provides the graphical results of an assay, showing thatentry by MBG, but not Ampho, luciferase virus into human osteosarcomacells is specifically inhibited by extracellular folic acid.

[0024]FIG. 12 provides the results of an assay showing that infection ofHOS cells by MBG, but not VSV, luciferase virus specifically decreasesin the presence of soluble bovine FBP.

[0025] FIGS. 13A-D provide the graphical results of assays showing thatexpression of FR-α in Jurkat-EctR cells reconstitutes permissivity toentry mediated by either MBG or EBO-Z GP. FIG. 13A shows thatpre-treatment of HeLa cells with phospholipase C resulted in completeabrogation of entry by EBO-Z luciferase virus. FIG. 13B shows that thereconstituted Jurkat-EctR cell clone A7-1, selected after challenge byEBO-Z-blasti virus and transduced with a cDNA encoding FR-α, isinfectable by both MBG and EBO-Z luciferase viruses, unlike Jurkat-EctRparental cells. FIG. 13C shows that Jurkat-EctR F10 cells reconstitutedfor infection by MBG-blasti virus and transduced with a cDNA encoding atruncated FR-α, are infectable by both MBG and EBO-Z luciferase viruses,unlike Jurkat-EctR parental cells. FIG. 13D shows that entry by EBO-Zluciferase virus into Jurkat-EctR F10 cells is specifically abrogated inthe presence of monoclonal mouse anti-FR IgG1 compared with levelsobserved with isotype anti-p24 IgG1.

[0026] FIGS. 14A-C provide the graphical results of assays showing thatinhibition of virion access to FR-α inhibits entry of EBO-Z luciferasevirus in naturally infectable HOS and Vero E6 cells. FIG. 14A shows thatentry by EBO-Z, but not Ampho, luciferase virus into HOS cells isspecifically inhibited by extracellular folic acid. FIG. 14B shows thatinfection of HOS cells by EBO-Z, but not VSV, luciferase virusspecifically decreases in the presence of soluble bovine FBP. FIG. 14Cshows that entry by EBO-Z, but not VSV, luciferase virus into Vero E6cells is completely abrogated in the presence of polyclonal rabbitanti-bovine FBP, compared with levels observed in normal rabbit sera.

DEFINITIONS

[0027] By “subject” or “individual” or “host” or “patient,” which termsare used interchangeably herein, is meant any subject, particularly amammalian subject, for whom diagnosis or therapy is desired,particularly humans. Other subjects may include non-human primates,cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

[0028] As used herein, the term “a filovirus-mediated disease condition”encompasses a condition which is a direct result of filovirus infection;and a condition which is an indirect result, e.g., a sequela, of afilovirus infection. Such conditions include, but are not limited to,fever, internal hemorrhaging, edema, organ failure, headache, malaise,myalgia, nausea, vomiting, bleeding of needle puncture sites,hematemesis, melena, petechiae, ecchymosis, maculopapular rash,disseminated intravascular coagulation, shock, jaundice, conjunctivitis,diarrhea, pharyngitis, convulsions, delirium, coma, oligura, andepistaxis.

[0029] As used herein, “an immune response” is meant to encompasscellular and/or humoral immune responses that are sufficient to inhibitor prevent infection, or prevent or inhibit onset of disease symptomscaused by a filovirus, and to reduce the likelihood of an infection by afilovirus.

[0030] The terms “peptide,” “oligopeptide,” “polypeptide,”“polyprotein,” and “protein”, are used interchangeably herein, and referto a polymeric form of amino acids of any length, which can includecoded and non-coded amino acids, chemically or biochemically modified orderivatized amino acids, and polypeptides having modified peptidebackbones. The term includes fusion proteins, including, but not limitedto, fusion proteins with a heterologous amino acid sequence, fusionswith heterologous and homologous leader sequences, with or withoutN-terminal methionine residues; immunologically tagged proteins; and thelike.

[0031] A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same whencomparing the two sequences. Sequence similarity can be determined in anumber of different manners. To determine sequence identity, sequencescan be aligned using the methods and computer programs, including BLAST,available over the world wide web at http://ww.ncbi.nlm.nih.gov/BLAST/.Another alignment algorithm is FASTA, available in the GeneticsComputing Group (GCG) package, from Madison, Wis., USA, a wholly ownedsubsidiary of Oxford Molecular Group, Inc. Other techniques foralignment are described in Methods in Enzymology, vol. 266: ComputerMethods for Macromolecular Sequence Analysis (1996), ed. Doolittle,Academic Press, Inc., a division of Harcourt Brace & Co., San Diego,Calif., USA. Of particular interest are alignment programs that permitgaps in the sequence. The Smith-Waterman is one type of algorithm thatpermits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187(1997). Also, the GAP program using the Needleman and Wunsch alignmentmethod can be utilized to align sequences. See J. Mol. Biol. 48: 443-453(1970).

[0032] Of interest is the BestFit program using the local homologyalgorithm of Smith Waterman (Advances in Applied Mathematics 2: 482-489(1981) to determine sequence identity. The gap generation penalty willgenerally range from 1 to 5, usually 2 to 4 and in many embodiments willbe 3. The gap extension penalty will generally range from about 0.01 to0.20 and in many instances will be 0.10. The program has defaultparameters determined by the sequences inputted to be compared.Preferably, the sequence identity is determined using the defaultparameters determined by the program. This program is available alsofrom Genetics Computing Group (GCG) package, from Madison, Wis., USA.

[0033] Another program of interest is the FastDB algorithm. FastDB isdescribed in Current Methods in Sequence Comparison and Analysis,Macromolecule Sequencing and Synthesis, Selected Methods andApplications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequenceidentity is calculated by FastDB based upon the following parameters:Mismatch Penalty:  1.00; Gap Penalty:  1.00; Gap Size Penalty:  0.33;and Joining Penalty: 30.0.

[0034] One parameter for determining percent sequence identity is the“percentage of the alignment region length” where the strongestalignment is found.

[0035] The percentage of the alignment region length is calculated bycounting the number of residues of the individual sequence found in theregion of strongest alignment. This number is divided by the totalresidue length of the target or query polynucleotide sequence to find apercentage. An example is shown below:

[0036] Target sequence:

[0037] Query sequence: GCGCGAAATACTCACTCGAGG     |   ||| |||| |||TATAGCCCTAC.CACTAGAGTCC 1    5    10     15

[0038] The region of alignment begins at residue 9 and ends at residue19. The total length of the target sequence is 20 residues. The percentof the alignment region length is 11 divided by 20 or 55%, for example.

[0039] Percent sequence identity is calculated by counting the number ofresidue matches between the target and query polynucleotide sequence anddividing total number of matches by the number of residues of the targetor query sequence found in the region of strongest alignment. For theexample above, the percent identity would be 10 matches divided by 11residues, or approximately, 90.9%

[0040] The percent of the alignment region length is typically at leastabout 55% of total length of the sequence, more typically at least about58%, and even more typically at least about 60% of the total residuelength of the sequence. Usually, percent length of the alignment regioncan be as great as about 62%, more usually as great as about 64% andeven more usually as great as about 66%.

[0041] The terms “antigen” and “epitope” are well understood in the artand refer to the portion of a macromolecule which is specificallyrecognized by a component of the immune system, e.g., an antibody or aT-cell antigen receptor. Epitopes are recognized by antibodies insolution, e.g., free from other molecules. Epitopes are recognized byT-cell antigen receptor when the epitope is associated with a class I orclass II major histocompatibility complex molecule.

[0042] “Antibody specificity”, in the context of antibody-antigeninteractions, is a term well understood in the art, and indicates that agiven antibody binds to a given antigen, wherein the binding can beinhibited by that antigen or an epitope thereof which is recognized bythe antibody, and does not substantially bind to unrelated antigens.Methods of determining specific antibody binding are well known to thoseskilled in the art, and can be used to determine the specificity ofantibodies of the invention for a FRα polypeptide.

[0043] The term “binds specifically,” in the context of antibodybinding, refers to high avidity and/or high affinity binding of anantibody to a specific polypeptide i.e., epitope of an FR polypeptide.Antibody binding to an epitope on a specific FR polypeptide (alsoreferred to herein as “an FR epitope”) is preferably stronger thanbinding of the same antibody to any other epitope, particularly thosewhich may be present in molecules in association with, or in the samesample, as the specific polypeptide of interest, e.g., binds morestrongly to a specific FR epitope than to a different FR epitope so thatby adjusting binding conditions the antibody binds almost exclusively tothe specific FR epitope and not to any other FR epitope, and not to anyother FR polypeptide which does not comprise the epitope. Antibodieswhich bind specifically to an FR polypeptide may be capable of bindingother polypeptides at a weak, yet detectable, level (e.g., 10% or lessof the binding shown to the polypeptide of interest). Such weak binding,or background binding, is readily discernible from the specific antibodybinding to the compound or polypeptide of interest, e.g. by use ofappropriate controls. In general, antibodies of the invention which bindto a specific FR polypeptide with a binding affinity of 10⁷ mole/l ormore, preferably 10⁸ mole/liters or more are said to bind specificallyto the specific FR polypeptide. In general, an antibody with a bindingaffinity of 10⁶ mole/liters or less is not useful in that it will notbind an antigen at a detectable level using conventional methodologycurrently used.

[0044] By “antisense polynucleotide” is meant a polynucleotide having anucleotide sequence complementary to a given polynucleotide sequence(e.g, a polynucleotide sequence encoding a FR polypeptide) includingpolynucleotide sequences associated with the transcription ortranslation of the given polynucleotide sequence (e.g., a promoter of apolynucleotide encoding FR polypeptide), where the antisensepolynucleotide is capable of hybridizing to a FR polypeptide-encodingpolynucleotide sequence. Of particular interest are antisensepolynucleotides capable of inhibiting transcription and/or translationof a FR-encoding polynucleotide either in vitro or in vivo.

[0045] By “transformation” is meant a permanent or transient geneticchange induced in a cell following incorporation of new DNA (i.e., DNAexogenous to the cell). Genetic change can be accomplished either byincorporation of the new DNA into the genome of the host cell, or bytransient or stable maintenance of the new DNA as an episomal element.Where the cell is a mammalian cell, a permanent genetic change isgenerally achieved by introduction of the DNA into the genome of thecell.

[0046] By “construct” is meant a recombinant nucleic acid, generallyrecombinant DNA, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences.

[0047] As used herein the term “isolated” is meant to describe acompound of interest (e.g., a virus, a peptide, etc.) that is in anenvironment different from that in which the compound naturally occurs.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified.

[0048] As used herein, the term “substantially purified” refers to acompound that is removed from its natural environment and is at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which it is naturally associated.

[0049] As used herein, the terms “treatment”, “treating”, and the like,refer to obtaining a desired pharmacologic and/or physiologic effect.The effect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease, or which may be susceptible to thedisease, but has not yet been diagnosed as having it (e.g., where thesubject is susceptible to infection by a pathogen, but has not yet beeninfected by the pathogen), including, but not limited to, reducing therisk of disease and/or death following infection by a filovirus;reducing the incidence of disease and/or death following infection by afilovirus; reducing the incidence or risk of infection by a filovirus;and reducing the extent of disease following infection by a filovirus;(b) inhibiting the disease, i.e., arresting its development, slowing itsprogression; and (c) relieving the disease, i.e., causing regression ofthe disease.

[0050] A “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids and tissue samples.

[0051] The term “immunologically active” refers to the capability of anatural, recombinant or synthetic FR polypeptide, or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies. As used herein, “antigenicamino acid sequence” means an amino acid sequence that, either alone orin association with a carrier molecule, can elicit an antibody responsein a mammal.

[0052] The term “mimetic,” as used herein, refers to a non-naturalcompound which exhibits one or more properties of a naturally occurringcompound.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0053] Methods and compositions are provided for at least slowing theprogression of a filovirus mediated disease condition in a host. In thesubject methods, an effective amount of an agent that at least reducesthe amount of folate receptor-mediated filovirus cell entry isadministered to the host. The subject methods find use in both theprevention and treatment of filovirus associated disease conditions,including Marburg and Ebola-Zaire virus mediated disease conditions.

[0054] Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0055] In this specification and the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

[0056] Methods of Treating A Filovirus-Mediated Disease Condition

[0057] The present invention provides methods of treating afilovirus-mediated disease condition in an individual. The methodsgenerally comprise administering to an individual an effective amount ofan agent that reduces a level of folate receptor-mediated viral entryinto a cell in the individual. In some embodiments, the inventionprovides methods of inhibiting binding of a filovirus to an FR receptoron the surface of a susceptible cell. In other embodiments, theinvention provides methods for reducing a level of FR receptor on thesurface of a cell that normally expresses FR receptor on its cellsurface. In other embodiments, the invention provides methods formodulating an FR receptor on a surface of a cell such that filovirusbinding to the FR receptor is reduced.

[0058] As used herein, the term “filovirus” refers to any knownfilovirus, including but not limited to, Marburg virus and Ebola virus,and subtypes of any known filovirus. Four subtypes of Ebola virus havebeen identified: Ivory Coast, Sudan, Zaire, and Reston. The nucleotidesequence of the complete genome of Ebola virus is found under GenBankAccession number NC_(—)002549. The nucleotide sequence of the completegenome of Marburg virus is found under GenBank Accession No.NC_(—)001608.

[0059] It has now been found that filovirus entry into a susceptiblecell is mediated by a folate receptor (FR), e.g., folate receptor alpha(FRα). A “susceptible cell” (also referred to herein as a “permissivecell”) is therefore any eukaryotic cell which expresses a FR on its cellsurface such that a filovirus can bind to the cell surface FR and enterthe cell. In general, a susceptible cell is a primate cell, e.g., ahuman cell or a monkey cell. A “susceptible cell” includes, but is notlimited to, a susceptible in an animal, e.g., a primate; a susceptiblecell in an organ from the animal (e.g., an organ removed from theanimal); a susceptible cell in a biological sample derived from ananimal; a susceptible cell in in vitro culture, e.g., a cell isolatedfrom an animal; a susceptible cell which is made susceptible by virtueof having been transformed with a nucleic acid construct comprising anucleic acid sequence that encodes a FR, e.g. cell line which does notnormally express FR on its cell surface but which does so afterintroduction into the cell line of a construct which results inexpression of FR on the cell surface, as described in the Examples; anda susceptible cell which is a cell line that is naturally permissive tofilovirus infection, including, but not limited to, HeLa cells andVeroE6 cells.

[0060] Whether a cell is susceptible to infection by a filovirus can bedetermined by any known method, including those described in theExamples.

[0061] The methods generally comprise administering to an individual aneffective amount of an agent that reduces a level of folatereceptor-mediated viral entry into a susceptible cell in the individual.In some embodiments, an “effective amount” of an agent that reducesFR-mediated entry into a cell is one that reduces filovirus entry into asusceptible cell by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90, or at leastabout 100%, when compared to a susceptible cell in the absence of theagent. In other embodiments, an “effective amount” of an agent iscontacted with a population of susceptible cells, where an “effectiveamount” is an amount that reduces the proportion of cells in thepopulation that is infected by the filovirus by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90, or at least about 100%, when compared with thepopulation not contacted by the agent.

[0062] It has further been shown that filovirus entry into a susceptiblecell can be inhibited by inhibiting binding of a filovirus to a FR onthe surface of a susceptible cell. Accordingly, in some embodiments, amethod of treating a filovirus-mediated disease condition in anindividual comprises administering to an individual an effective amountof a substance that inhibits binding of a filovirus to a FR on thesurface of a susceptible cell in the individual. As used herein, theterm “agent” refers to any substance that inhibits binding of afilovirus to FR, including, but not limited to, antibody specific forFR; folic acid and its derivatives; and soluble FR. Generally, thesemethods involve blocking a binding event between a filovirus and an FR.The methods provide for inhibiting binding of a filovirus to an FR onthe surface of a susceptible cell by at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, or at least about 100%, when compared to the amount of filovirusthat binds to an FR on the surface of a control cell, e.g., a cell notcontacted with the agent.

[0063] In other embodiments, the invention provides methods for reducinga level of FR receptor on the surface of a cell that normally expressesFR receptor on its cell surface, comprising contacting the cell with aneffective amount of an agent that reduces a level of FR on the cellsurface. In these embodiments, an “effective amount” of an agent is anamount that is effective in reducing the level of FR on the cell surfaceby at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, or at least about 100%, whencompared to the level of FR expressed on the cell surface of a controlcell not contacted with the agent. The level of FR on the cell surfacecan be determined (e.g., measured) using any known method, including,but not limited to, contacting a cell with a detectably labeled antibodyspecific for FR, and determining the amount of bound antibody using,e.g., fluorescence activated cell sorting (FACS), radioligand binding,immunofluorescence, Northern (RNA) blotting, Western (protein) blotting,and in situ hybridization.

[0064] Reduction in a level of FR receptor displayed on the surface of acell that normally expresses FR receptor on its cell surface may beaccomplished by a variety of means, including, but not limited to,reducing the level of transcription of a gene encoding the FR; reducingthe level of FR-encoding mRNA available to be translated; reducing thelevel of translation of an FR-encoding mRNA; reducing formation of a GPIlinkage on the FR, thereby resulting in secretion of the FR from thecell, rather than placement in the cell membrane; reducing the rateand/or level of any cell biological process that normally results inexpression of FR on the cell surface. In some cases it may be preferablythat the method is specific or relatively specific to FR, e.g., themethod does not reduce the level of any other protein on the cellsurface.

[0065] Reduction in expression of an FR-encoding gene may beaccomplished by using antisense to the FR-encoding gene. Variousderivatives of the antisense sequence may be prepared, where thephosphates may be modified, where oxygens may be substituted with sulfurand nitrogen, the sugars may be modified, and the like. The antisensesequences may be used by themselves or in conjunction with various toxicmoieties, such as metal chelates, sensitizers, ribozymes, and the like.Antisense and/or ribozyme sequences may be used to inhibit FR geneexpression. Antisense polynucleotides, and methods of using such, aredescribed in numerous publications, including, e.g., “AntisenseTechnology: A Practical Approach” Lichtenstein and Nellen, eds. (1997)IRL Press.

[0066] Antisense molecules can be used to down-regulate expression ofFR-encoding genes in cells. The anti-sense reagent may be antisenseoligodeoxynucleotides (ODN), particularly synthetic ODN having chemicalmodifications from native nucleic acids, or nucleic acid constructs thatexpress such anti-sense molecules as RNA. The antisense sequence iscomplementary to the mRNA of the targeted gene, and inhibits expressionof the targeted gene products. Antisense molecules inhibit geneexpression through various mechanisms, e.g. by reducing the amount ofmRNA available for translation, through activation of RNAse H, or sterichindrance. One or a combination of antisense molecules may beadministered, where a combination may comprise two or more differentsequences.

[0067] Antisense molecules may be produced by expression of all or apart of the target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996) Nature Biotechnology 14:840-844).

[0068] A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

[0069] Antisense oligonucleotides may be chemically synthesized bymethods known in the art (see Wagner et al. (1993) supra.) Preferredoligonucleotides are chemically modified from the native phosphodiesterstructure, in order to increase their intracellular stability andbinding affinity. Such modifications have been previously discussed withrespect to the use of probes.

[0070] As an alternative to anti-sense inhibitors, catalytic nucleicacid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be usedto inhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995) Nucl. Acids Res 23:4434-42). Examples of oligonucleotideswith catalytic activity are described in WO 9506764. Conjugates ofanti-sense ODN with a metal complex, e.g. terpyridylCu(II), capable ofmediating mRNA hydrolysis are described in Bashkin et al. (1995) ApplBiochem Biotechnol 54:43-56.

[0071] In other embodiments, the invention provides methods formodulating an FR receptor on a surface of a cell such that filovirusbinding to the FR receptor is reduced. The agent may modulate theconfiguration of the surface membrane-associated FR such that it nolonger binds filovirus. Alternatively, the agent may be one thatmodulates trafficking, clustering, or internalization of FR. The agentmay be one that inhibits glycosylation of FR. Roberts et al. (1998)Arch. Biochem. Biophys. 351:227-235.

[0072] In any of the above-described methods of the invention fortreating a filovirus-mediated disease condition, more than one agent maybe administered to an individual in need of treatment. Thus, forexample, a mixture of two or more monoclonal antibodies specific fordistinct, non-overlapping epitopes on an FR may be administered to theindividual. Mixtures of two or more different agents, e.g., a monoclonalantibody specific for an FR and a folate receptor ligand, may also beadministered.

[0073] Routes of Administration

[0074] An agent is administered to an individual using any availablemethod and route suitable for drug delivery, including in vivo and exvivo methods, as well as systemic and localized routes ofadministration.

[0075] Conventional and pharmaceutically acceptable routes ofadministration include intranasal, intramuscular, intratracheal,intratumoral, subcutaneous, intradermal, topical application,intravenous, rectal, nasal, oral and other parenteral routes ofadministration. Routes of administration may be combined, if desired, oradjusted depending upon the agent used and/or the desired effect. Acomposition comprising an agent can be administered in a single dose orin multiple doses.

[0076] An agent can be administered to a host using any availableconventional methods and routes suitable for delivery of conventionaldrugs, including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

[0077] Parenteral routes of administration other than inhalationadministration include, but are not necessarily limited to, topical,transdermal, subcutaneous, intramuscular, intraorbital, intracapsular,intraspinal, intrasternal, and intravenous routes, i.e., any route ofadministration other than through the alimentary canal. Parenteraladministration can be carried to effect systemic or local delivery ofthe agent. Where systemic delivery is desired, administration typicallyinvolves invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

[0078] An agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

[0079] Methods of administration of an agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration.

[0080] The invention also contemplates opthalmic administration of anagent, which generally involves invasive or topical application of apharmaceutical preparation to the eye. Eye drops, topical cremes andinjectable liquids are all examples of suitable formulations fordelivering drugs to the eye.

[0081] Dosages

[0082] Although the dosage used will vary depending on the clinicalgoals to be achieved, a suitable dosage range is one which provides upto about 1 μg to about 1,000 μg or about 10,000 μg of agent can beadministered in a single dose. Alternatively, a target dosage of agentcan be considered to be about 1-10 μM in a sample of host blood drawnwithin the first 24-48 hours after administration of the agent.

[0083] In general, an agent is administered in an amount within therange from about 0.002 mg/kg to about 10 mg/kg, or from about 0.01 mg/kgto about 3 mg/kg body weight. Where an agent is to be administeredintravenously, it will be formulated in conventional vehicles, such asdistilled water, saline, Ringer's solution or other conventionalcarriers.

[0084] Methods for Immunizing a Host

[0085] The invention further provides methods for immunizing a hostagainst a filovirus mediated disease condition. In general, the methodsinvolve administering to a host an effective amount of an immunogen thatcauses said host to mount an immune response against membrane boundfolate receptors, where antibodies are generated that inhibit filovirusbinding to the FR, thereby reducing entry of the filovirus into apermissive cell. In some embodiments, the immunogen is a membrane boundfolate receptor or fragment thereof. Methods of generating an immuneresponse in a host are known in the art and need not be elaborated uponhere. Whether an individual has mounted an immune response to a membranebound folate receptor can be readily determined. For example, abiological sample, such as a blood or serum sample, is removed from theindividual, and the presence of antibodies that specifically bind tomembrane bound FR and block or inhibit filovirus binding to the FR isdetected using assays such as those described in the Examples.

[0086] Antibodies generated in a human in the manner described above maybe isolated and used prophylactically in a passive immunization protocolto protect another human against a filovirus-mediated disease condition.For example, a health care worker or other medical personnel or supportstaff who anticipate being in a situation which puts him/her at risk forexposure to filovirus may be treated prophylactically with anti-FRantibodies generated in another human. In some instances, e.g., wheremedical personnel is exposed to filovirus for a short period, e.g., 1-7days, or 1-4 weeks, short-term protection of such individuals, such asmay be afforded by passive immunization, may be adequate to protect theindividual from filovirus infection, or may reduce symptoms duringinfection (e.g., attenuation of disease).

[0087] Method for Identifying a Viral Cell Surface Receptor

[0088] The invention provides a method of a identifying a cell surfacereceptor used by a virus for entry into a cell. The methods generallycomprise: (a) identifying a cell line that is non-permissive for entryof the virus; (b) transfecting a population of said non-permissive cellline with a genomic or a cDNA library obtained from a cell linepermissive for entry of the virus; (c) identifying at least one cellfrom said transfected cell population which is permissive for entry ofthe virus; and (d) identifying at least one gene of the permissive cellline in the genome of the transfected permissive cell. These methods areuseful for identifying a cell surface receptor for a filovirus. Inparticular embodiments, a population of non-permissive cells istransfected with a cDNA library made from a permissive cell such that amember of the cDNA library are expressed in a cell of the transfectednon-permissive cell population. To determine which cell(s) of thetransfected non-permissive population are now permissive for filovirusinfection, a pseudotype virus may be employed, in which anenvelope-negative mutant virus carrying a selectable marker isengineered to contain a filovirus envelope glycoprotein. In a particularembodiment, a pseudovirus as described in the Examples is used.

[0089] Agents Effective for Treating a Filovirus-Mediated DiseaseCondition

[0090] The present invention provides agents for treating afilovirus-mediated disease condition. In some embodiments, an agent thatis suitable for treating a filovirus-mediated disease condition is onethat inhibits binding of a filovirus to an FRα on the surface of asusceptible cell. As used herein, “an FR antagonist” is any agent thatinhibits a binding event between a filovirus and a membrane-bound FR,including, but not limited to, a soluble FR, an antibody specific for anFR, a filovirus-derived FR ligand, an FR ligand, and a fragment,derivative, or mimetic of any of the foregoing. In other embodiments, anagent is one that modulates trafficking, clustering, or internalizationof membrane bound folate receptors. In other embodiments, an agent isone that modulates expression or configuration of a membrane-boundfolate receptor such that binding to a filovirus is reduced.

[0091] Folic Acid Receptor

[0092] FR is a protein encoded by a gene that is a member of the folatereceptor family. Members of this gene family have a high affinity forfolic acid and for several reduced folic acid derivatives, and mediatedelivery of 5-methyltetrahydrofolate to the interior of eukaryoticcells. The gene is composed of 7 exons: exons 1-4 encode the 5′untranslated region and exons 4 through 7 encode the open reading frame.Due to the presence of 2 promoters, multiple transcription start sites,and alternative splicing of exons, at least 8 transcript variants arederived from this gene. These variants differ in the length of 5′ and 3′UTR, but they encode an identical amino acid sequence. Elwood et al.(1997) Biochem. 36: 1467-1478.

[0093] Human FRα (also referred to as folate binding protein) issynthesized in cells as an integral membrane-associated protein and as asoluble protein. Sadavisan and Rothenberg (1989) J. Biol. Chem.264:5806-5811. The amino acid sequence of human FRα is provided underGenBank Accession No. NM_(—)016731. The membrane-associated form is aglycosyl phosphatidyl inositol linked protein, while the secreted formlacks the GPI moiety. Nucleotide and amino acid sequences of FR fromother species are also publicly available under GenBank.

[0094] As used herein, the terms “folate receptor ” and “folate bindingprotein” are used interchangeably herein and refer to FR from any of avariety of species, including, but not limited to, human, murine (mouseor rat), bovine, or other mammalian species. In some embodiments, an FRis a human FRα having the amino acid sequence set forth in GenBankAccession No. NM_(—)016731. In some embodiments, an FR is a polypeptidecomprising an amino acid sequence that shares at least about 50%, atleast about 60%, at least about 75%, at least about 80%, at least about90%, or at least about 95% or more sequence identity with the sequenceset forth in GenBank NM_(—)016731. It is generally preferred that the FRadministered to the individual does not elicit an immune reaction in theindividual to the FR. “FR” also encompasses fragments of an FR thatinhibit binding of a filovirus to an FR on the surface of a susceptiblecell. Full length FR is a protein of about 226 amino acids. In someembodiments, FR is a fragment of from about 15 to about 20, from about20 to about 25, from about 25 to about 50, from about 50 to about 75,from about 75 to about 100, from about 100 to about 125, from about 125to about 150, from about 150 to about 175, or from about 175 to about200 amino acid, up to the full length protein. “FR” also encompassesfusion proteins, wherein an FR is fused in-frame to a fusion partnerwhich is a heterologous protein, i.e., a non-FR protein. Fusion partnersinclude, but are not limited to, immunological tags, cytokines, carrierproteins (e.g., albumin), antibody (including antibody fragments),cytotoxic agents, protein domains that cause multimerization, and thelike.

[0095] “FR” may be an FR isolated from a source in which it naturallyoccurs, may be a synthetic FR, or may be an FR made recombinantly. “FR”also encompasses mimetics of a naturally occurring FR, and peptoids.Methods of synthesizing peptoids, and peptoid libraries, and methods ofscreening same are found in, e.g., U.S. Pat. Nos. 5,965,695; and6,075,121.

[0096] FR may be made recombinantly using standard techniques ofmolecular biology; may be made synthetically using standard techniquesof protein synthesis; may be isolated from a source in which itnaturally occurs (e.g., milk or other body fluids); or a combination ofany of the foregoing. FR polypeptides can be isolated from a biologicalsource, using affinity chromatography, e.g., using antibodies specificfor FR which are immobilized on a solid support.

[0097] The polypeptides may be expressed in prokaryotes or eukaryotes inaccordance with conventional ways, depending upon the purpose forexpression. For large scale production of the protein, a unicellularorganism, such as E. coli, B. subtilis, S. cerevisiae, insect cells incombination with baculovirus vectors, or cells of a higher organism suchas vertebrates, particularly mammals, e.g. COS 7 cells, CHO cells,HEK293 cells, HeLa cells, and the like, may be used as the expressionhost cells. In some situations, it is desirable to express the gene ineukaryotic cells, where the protein will benefit from native folding andpost-translational modifications. The polypeptide can then be isolatedfrom cell culture supernatant or from cell lysates using affinitychromatography methods or anion exchange/size exclusion chromatographymethods, as described above.

[0098] One may employ solid phase peptide synthesis techniques, wheresuch techniques are known to those of skill in the art. See Jones, TheChemical Synthesis of Peptides (Clarendon Press, Oxford)(1994).Generally, in such methods a peptide is produced through the sequentialadditional of activated monomeric units to a solid phase bound growingpeptide chain.

[0099] With the availability of the protein or fragments thereof inlarge amounts, by employing an expression host, the protein may beisolated and purified in accordance with conventional ways. A lysate maybe prepared of the expression host and the lysate purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique.

[0100] For use in the methods of the invention, an FR may beadministered in a formulation in association with (e.g., chemicallyassociated, or in admixture with) another macromolecule, including, butnot limited to, a protein, including, but not limited to, albumin; ananoparticle; a lipid, including, but not limited to liposomes; apolysaccharide; a polyalcohol, including, but not limited to, apolyethylene glycol; a glycoprotein; and combinations of the foregoing.

[0101] Folate Receptor Ligands

[0102] Agents that inhibit binding of a filovirus to a membrane bound FRalso include folate receptor ligands, and derivatives and mimeticsthereof. A folate receptor ligand may be folic acid (5-methyltetrahydrofolic acid); or a derivative thereof, including, but notlimited to, dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate,5,10-methylenetetrahydrofolate, 5,10-methenyltetrahydrofolate,5,10-formiminotetrahydrofolate, 5-formyltetrahydrofolate (leucovorin),and 10-formyltetrahydrofolate.

[0103] Filovirus-derived Folate Receptor Ligands

[0104] Agents that inhibit binding of a filovirus to a membrane-bound FRfurther include filovirus molecules that bind to FR, which reducebinding of a filovirus to the FR, and which therefore reduce FR-mediatedfilovirus entry into a permissive cell. Filovirus-derived folatereceptor ligands include, but are not limited to, a filovirus envelopeglycoprotein. Marburg envelope glycoprotein is described in Xu et al.(1998) Nat. Med. 4: 37-42; and Ebola virus Zaire envelope glycoproteinis described in GenBank accession number U31033. “Filovirus envelopeglycoprotein,” as used herein in the context of an agent that inhibitsbinding of a filovirus to a membrane-bound FR, encompasses full-lengthfilovirus envelope glycoprotein (GP); fragments of a filovirus envelopeGP which mediate binding to an FR; fusion proteins comprising thefilovirus envelope GP (or fragment thereof. Those skilled in the art,using any known method, including those described herein, can readilydetermine any fragment of a filovirus envelope glycoprotein that caninhibit binding of a filovirus to a membrane-bound FR.

[0105] Antibodies

[0106] An agent that inhibits binding of a filovirus to an FR on thesurface of a susceptible cell may be an antibody. As used herein, theterm “antibodies” includes antibodies of any isotype, fragments ofantibodies which retain specific binding to antigen, including, but notlimited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies,humanized antibodies, single-chain antibodies, and fusion proteinscomprising an antigen-binding portion of an antibody and a non-antibodyprotein. The antibodies may be detectably labeled, e.g., with aradioisotope, an enzyme that generates a detectable product, a greenfluorescent protein, and the like. The antibodies may be furtherconjugated to other moieties, such as members of specific binding pairs,e.g., biotin (member of biotin-avidin specific binding pair), and thelike.

[0107] Methods of generating an antibody to an FR are well known in theart and need not be elaborated upon herein. Such methods are found in avariety of standard textbooks, such as Antibodies: A Laboratory Manual,E. Harlow and D. Lane, eds. 1988, Cold Spring Harbor Laboratory Press.Antibodies generated to an FR may be screened for the ability to inhibitbinding of a filovirus to an FR, using, e.g., the methods described inthe Examples.

[0108] Agent that Modulate Trafficking, Clustering, or Internalizationof Membrane Bound Folate Receptors

[0109] Agents that modulate trafficking, clustering, or internalizationof membrane-bound FR include, but are not limited to, an agent thatinhibits or interferes with glycolipid anchors, e.g., phospholipase-C;an agent that inhibits or interferes with glycosylation of an FR; agentsthat disrupt the pathway to construct GPI anchors in the endoplasmicreticulum; agents that disrupt acidification of vesicles; agents thatinhibit recycling of GPI-linked proteins such as FR; agents that preventmultimerization of FR at the cell surface (e.g., FR fragments that bindto multimerization domains; and agents that prevent endocytosis of FR orGPI-linked proteins.

[0110] Agents that Modulate Expression or Configuration of aMembrane-bound Folate Receptor such that Binding to a Filovirus isReduced

[0111] Agents that modulate expression or configuration of amembrane-bound folate receptor such that binding to a filovirus isreduced include, but are not limited to, antisense molecules (asdiscussed above); ribozymes (as discussed above); compounds thatselectively reduce transcription of a FR gene; and dominant-negativeforms of FR which reduce and/or prevent proper binding, folding, ormultimerization of FR on the cell surface.

[0112] Compositions

[0113] The present invention further provides compositions comprising anagent of the invention. These compositions may include a buffer, whichis selected according to the desired use of the agent, and may alsoinclude other substances appropriate to the intended use. Those skilledin the art can readily select an appropriate buffer, a wide variety ofwhich are known in the art, suitable for an intended use. In someinstances, the composition can comprise a pharmaceutically acceptableexcipient, a variety of which are known in the art and need not bediscussed in detail herein. Pharmaceutically acceptable excipients havebeen amply described in a variety of publications, including, forexample, A. Gennaro (1995) “Remington: The Science and Practice ofPharmacy”, 19th edition, Lippincott, Williams, & Wilkins.

[0114] Pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

[0115] Screening Assays

[0116] The present invention provides methods of screening for candidateagents that are useful in treating a filovirus-mediated diseasecondition. In some embodiments, methods are provided for screening forcandidate agents that inhibit binding of a filovirus to a FR on thesurface of a susceptible cell. In other embodiments, methods areprovided for screening for candidate agents that reduce a level of FR onthe surface of a susceptible cell.

[0117] The term “candidate agent” is used interchangeably herein withthe terms “candidate substance” and “candidate compound”. A “candidateagent,” as used herein, describes any molecule, e.g. protein; peptide;natural or synthetic inorganic or organic compound, or pharmaceutical,with the capability of reducing filovirus entry into a susceptible cell,as described above. Generally a plurality of assay mixtures is run inparallel with different agent concentrations to obtain a differentialresponse to the various concentrations. Typically, one of theseconcentrations serves as a negative control, i.e. at zero concentrationor below the level of detection.

[0118] Candidate agents encompass numerous chemical classes, and may benatural or synthetic inorganic or organic molecules, which may be smallorganic compounds having a molecular weight of more than 50 and lessthan about 2,500 daltons. Candidate agents may comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

[0119] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, glycosylation, amidification,etc. to produce structural analogs.

[0120] Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

[0121] A variety of other reagents may be included in the screeningassay. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc that are used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used. The mixture of components are added in any order thatprovides for the requisite binding. Incubations are performed at anysuitable temperature, typically between 4 and 40° C. Incubation periodsare selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 and 1hours will be sufficient.

[0122] Methods of Identifying Candidate Agents that Inhibit FilovirusBinding to FR

[0123] In some embodiments, methods are provided for identifying acandidate agent that inhibits filovirus binding to FR. In some of theseembodiments, the methods are cell-free methods. In other embodiments,the methods are cell-based methods. In general, the methods describedbelow are in vitro screening methods. Candidate agents identified by themethods described below include those that act to block binding of afilovirus to an FR; and those that act to modulate a configuration of anFR such that filovirus binding is reduced. Candidate agents of interestare those that reduce filovirus entry into a cell. Accordingly, in someembodiments, the methods provide for identifying a candidate agent thatreduces filovirus entry into a cell.

[0124] As used herein, “determining” includes “measuring” and“detecting,” e.g., the determination may be quantitative orsemi-quantitative (e.g., “measuring”) or qualitative (e.g.,“detecting”).

[0125] Agents which decrease FR-filovirus binding to the desired extentmay be selected for further study, and assessed for cellularavailability, cytotoxicity, biocompatibility, etc.

[0126] Of interest are candidate agents that inhibit FR-filovirusbinding by at least about 10%, at least about 15%, at least about 20%,at least about 25%, more preferably at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 50%, atleast about 100%, or 2-fold, at least about 5-fold, or at least about10-fold or more when compared to a suitable control. A candidate agentwhich inhibits FR-filovirus binding can also be one that abrogatesmeasurable FR-filovirus binding completely.

[0127] Also of interest are candidate agents that reduce filovirus entryinto a cell susceptible to filovirus infection by at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about50%, at least about 100%, or 2-fold, at least about 5-fold, or at leastabout 10-fold or more when compared to a suitable control. A candidateagent which inhibits FR-filovirus entry can also be one that abrogatesmeasurable FR-filovirus entry completely.

[0128] Also of interest are candidate agents that reduce the number ofcells in a population of susceptible cells that are infected byfilovirus by at least about 10%, at least about 15%, at least about 20%,at least about 25%, at least about 50%, or at least about 100%, whencompared to a suitable control.

[0129] Cell-free Methods

[0130] A cell-free method to identify candidate agents that inhibitbinding of a filovirus to an FR generally comprise:

[0131] a) contacting a candidate agent with a sample containing an FRand a filovirus; and

[0132] b) determining whether binding between the FR and the filovirusis reduced.

[0133] In some embodiments, the screening methods will employ afilovirus envelope glycoprotein. Thus, the term “filovirus,” in thecontext of screening assays of the invention, encompasses a filovirusenvelope glycoprotein. Thus, in these embodiments, the methods comprisecontacting a candidate agent with a sample containing an FR and afilovirus envelope glycoprotein; and determining whether FR-filovirusenvelope glycoprotein binding is reduced, compared to binding in theabsence of the candidate agent.

[0134] Marburg envelope glycoprotein is described in Xu et al. (1998)Nat. Med. 4: 37-42; and Ebola virus Zaire envelope glycoprotein isdescribed in GenBank accession number U31033. “Filovirus envelopeglycoprotein,” as used herein, encompasses full-length filovirusenvelope glycoprotein (GP); fragments of a filovirus envelope GP whichmediate binding to an FR; fusion proteins comprising the filovirusenvelope GP (or fragment thereof), including, but not limited to,epitope-tagged filovirus envelope GP. In addition, the filovirusenvelope GP may be detectably labeled.

[0135] Determining whether FR-filovirus binding is reduced can beaccomplished in a variety of ways, including, but not limited to, anyknown immunological assay method, including, but not limited to, animmunological assay in which FR-filovirus binding is detected usingantibody to the FR, to the filovirus (where the antibody is not one thatinhibits FR-filovirus binding), to an epitope tag moiety of an FR fusionprotein, or to an epitope tag moiety of a filovirus envelope GP; anenzyme-linked immunological assay; an immunological assay in which theFR and/or the filovirus is detectably labeled.

[0136] Cell-based Assays

[0137] In some embodiments, the screening assays are cell-based assays.In some of these embodiments, the methods generally comprise contactinga cell susceptible to infection by a filovirus with a candidate agent;and determining an effect, if any, on filovirus binding to the cell. Inother of these embodiments, the methods generally comprise contacting acell susceptible to infection by a filovirus with a candidate agent; anddetermining an effect, if any, on filovirus entry into the cell.

[0138] Cells suitable for use in these methods are any permissiveeukaryotic cell, including cells that are naturally permissive,including, but not limited to, those cells shown in FIG. 2 that wereshown to be permissive for filovirus infection (e.g., HOS, HeLa, VeroE6,CHO; and cells that are modified to be permissive for filovirus entry,including, but not limited to, cells as described in the Examples.

[0139] In some embodiments, a pseudotype virus, e.g., an HIV-1 virusengineered to contain a nucleotide sequence encoding a filovirusenvelope glycoprotein, such as described in the Examples, may beemployed. Thus, in the context of a cell-based screening assay of theinvention, “filovirus” encompasses pseudotype virus comprising anucleotide sequence encoding a filovirus envelope glycoprotein.

[0140] Whether filovirus binds to and enters the cell can be determinedusing any known assay method, including, but not limited to, assaysdescribed in the Examples. As described in the Examples, a pseudotypevirus can be engineered to comprise a filovirus envelope GP and anucleotide sequence encoding a selectable marker, or a detectable geneproduct (e.g., luciferase, GFP, and the like). Filovirus entry into thecell can then be determined by, e.g., measuring the amount of detectablegene product in the cell. The assay can also be conducted so as todetermine the number of cells in a cell population that are infected.

[0141] Methods of Screening for Candidate Agents that Reduce a Level ofFR on the Surface of a Susceptible Cell

[0142] In general, methods for reducing a level of FR on the surface ofa susceptible cell comprise contacting a susceptible cell with acandidate agent; and determining the effect, if any, on the level of FRon the surface of the cell.

[0143] Cells which may be employed are as described above, and aregenerally cells that are susceptible to filovirus infection by virtue ofexpressing an FR on the cell surface. Methods for determining whether acandidate agent reduces a level of FR on the cell surface includemethods of measuring or detecting filovirus entry into the cell, asdescribed above.

[0144] Determination of whether a candidate agent reduces FR geneexpression can be carried out using any known assay. For example, apolymerase chain reaction (PCR) can be carried out on mRNA isolated froma cell, using a primer pair specific for an FR-encoding transcript. AcDNA copy of the population of isolated mRNA, and PCR performed on thecDNA. Another method which may be employed involves an assay in which acell is transfected with a construct comprising an FR promoter drivingtranscription of a reporter gene, and the effect of the candidate agenton the level of transcription of the reporter gene is determined.

[0145] The following examples are offered by way of illustration and notby way of limitation. The following examples are put forth so as toprovide those of ordinary skill in the art with a complete disclosureand description of how to make and use the present invention, and arenot intended to limit the scope of what the inventors regard as theirinvention nor are they intended to represent that the experiments beloware all or the only experiments performed. Efforts have been made toensure accuracy with respect to numbers used (e.g. amounts, temperature,etc.) but some experimental errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, molecularweight is weight average molecular weight, temperature is in degreesCelcius, and pressure is at or near atmospheric.

EXPERIMENTAL Example 1

[0146] Identification of a Cofactor for Entry of Marburg and EbolaViruses into Susceptible Cells

[0147] Materials and Methods

[0148] Cell lines. Human osteosarcoma (HOS), HeLa, 293T, Vero E6, andCHO-K1 cells were cultured as recommended by the American Type CultureCollection (ATCC). Jurkat T-cells stably expressing the ecotropic murineleukemia virus (MLV) receptor (Jurkat-EctR, kindly provided by Dr. G.Nolan, Stanford University) were cultured as recommended for JurkatT-cells by the ATCC. The NIH-3T3 based MLV packaging cell line PT67(Clontech, Palo Alto, Calif.) was cultured as previously described(Miller and Chen (1996) J. Virol. 70, 5564-5571. Human osteosarcoma(GHOST) indicator cell lines carrying a human green fluorescent protein(GFP) reporter gene driven by an HIV-2 Tat-dependent LTR (provided byDr. D. Littman, Skirball Institute) were cultured as previouslydescribed (Trkola et al., (1998) J. Virol. 72, 1876-1885).

[0149] Plasmids and cDNA library amplification The molecular clonepNL-Luc-E⁻R⁻(Connor et al., (1995) Virology 206, 935-944), the HIV-1NL4-3 provirus carrying a luciferase reporter gene driven by the 5′ LTR(along with mutations in env, nef, and vpr), was a gift of Dr. N. Landau(Salk Institute) via the AIDS Research and Reference Reagent Program.HIV-blasti, an HIV-1 proviral construct carrying the blasticidin Sdeaminase gene driven * by the 5′ LTR along with null mutations in envand nef was provided by Dr. R. Sutton (Baylor University). Mammalianexpression plasmid pVSV-G encoding the vesicular stomatitis virus-G(VSV-G) protein was provided by Dr. J. Burns (University of California,San Diego), and pMULV-A encoding the amphotropic (Ampho) murine leukemiavirus (MLV) env (Landau et al., (1991) J. Virol. 65, 162-169) wasprovided by Dr. K. Page (University of California, San Francisco). ThecDNA clones encoding MBG GP and EBO-Z GP were provided by Dr. A. Sanchez(Centers for Disease Control and Prevention) and cloned into thepCMV4neo expression vector (Goldsmith et al., (1994) J. Biol. Chem. 269,14698-14704) as previously described (Chan et al., (2000) J. Virol. 74,4933-4937). The full length and 3′ truncated cDNA (E4-1) encoding FR-αwas recovered by PCR from the HeLa retroviral library as describedbelow, ligated by TA cloning into pCR2.1, and subcloned into themammalian expression vector. The bicistronic mammalian expression vectorpIRES2-EGFP (Clontech, Palo Alto, Calif.) was used to assesstransfection efficiency in Jurkat-EctR cells.

[0150] A bacterial glycerol stock transformed with the MLV retroviralcDNA library (pLib MLV backbone) derived from HeLa cells (2×10⁶independent clones) was plated on LB agar (100 μg/ml ampicillin) platesas described by the manufacturer (Clontech, Palo Alto, Calif.).Approximately 8×10⁶ bacterial colonies were amplified in LB liquidbroth, and library DNA was extracted for subsequent virus packaging andtransduction into Jurkat-EctR cells. The plasmid pLib-GFP encoding thegreen fluorescent protein in the MLV backbone was used as a marker forquantitating efficiency of transduction.

[0151] Antibodies. For detection of wildtype MBG virus infection,convalescent guinea pig antisera to MBG virus (Musoke) was generated.FITC-conjugated anti-guinea pig secondary antisera was purchased. Forinhibition of pseudotype virus entry, polyclonal rabbit antisera raisedagainst FBP in bovine milk (Biogenesis, Poole, England, UK) and normalrabbit sera (kindly provided by Dr. 0. Keppler, Gladstone Institute ofVirology and Immunology, CA) were compared. Similarly, monoclonal mouseIgG1 ascites raised against human FR-α (kindly provided by Dr. W.Franklin, University of Colorado Health Sciences Center) and monoclonalmouse IgG1 ascites raised against HIV-1 Gag p24 antigen were compared.For assessing binding of FBP to CHO-K1 cells expressing virus GP byimmunofluorescence, polyclonal goat anti-bovine FBP antibody andfluorescein-conjugated rabbit anti-goat IgG Fc were used for staining(Rockland, Gilbertsville, Pa.).

[0152] Preparation of pseudotype virus stocks. To prepare HIV-1pseudotype virus carrying the luciferase gene (Luc⁺) with the Ampho,VSV-G, MBG, or EBO-Z GP, pNL-Luc-E⁻R⁻ (2 μg/well) was co-transfectedwith each envelope glycoprotein expression vector (2 μg/well) using theCaPO₄ method in 293T cells as previously described (Chan et al., (1999)J. Virol. 73, 2350-2358). To prepare selectable HIV-1 pseudotype viruscarrying the blasticidin S deaminase gene (blasti), 293T cells (400,000cells/well in 6-well plates) were transiently co-transfected using theCaPO₄ method with pHIV-blasti (2 μg/well) and an envelope GP expressionvector encoding MBG GP (2 μg/well), EBO-Z GP (2 μg/well), or VSV-G GP (2μg/well) as above. Viral stocks were sterile filtered (0.2 μm) andharvested after 36 h in a BSL3 facility.

[0153] Protocol for genetic reconstitution of permissivity to filovirusentry. To reconstitute permissivity for MBG virus entry, library DNA (3μg/well) or pLib-GFP (3 μg/well) reporter plasmid was packaged intopseudovirions by transiently co-transfecting PT67 packaging cells(300,000 cells/well in 6-well plates) along with pVSV-G (1 μg/ml) usingthe CaPO₄ method and harvesting culture supernatants on day 2post-transfection. Subsequently, approximately 1.2×10⁸ Jurkat-EctR cellswere transduced with library-containing viral supernatants via spininfection (1.3×10⁶ RPM, 32° C., 2 h) in 6 separate batches. In parallel,Jurkat-EctR cells were transduced with pseudovirions carrying pLib GFP.By quantitating GFP-positive Jurkat-EctR cells two days aftertransduction, library infection was optimized to achieve reproducible30-40% transduction efficiency. Two days later, selectable MBG-blasti orVSV-blasti pseudotype virus was harvested and used to challenge parentalcells or cells transduced with the library. In parallel, GHOST cells(250,000 cells/well) were inoculated with 1 ml of selectable pseudotypeHIV virus, since they are permissive to entry by MBG, EBO-Z, and VSVviruses (Chan et al., (2000) J. Virol. 74, 4933-4937). GHOST cellsexpress the GFP reporter only in the presence of HIV-1 Tat protein(Trkola et al., (1998) J. Virol. 72, 1876-1885), and thus aftersuccessful infection by HIV-1 pseudotype virions. Therefore, two daysfollowing challenge, by quantitating the percent of GFP-positive GHOSTcells as previously described (Chan et al., (2000) J. Virol. 74,4933-4937), relative levels of active MBG-blasti and VSV-blasti viruseswere estimated as the percent of permissive cells that are infectedusing a given virus stock. When virus inocula achieved entry in morethan 50% of the GHOST cell culture, the transduced Jurkat-EctR cellschallenged with the same virus stock were transferred into mediumcontaining blasticidin S (ICN, 40 μg/ml). After selection for 2 weeks,cells were monitored for viability by Trypan blue exclusion and countedon a hemacytometer. Selected cells were expanded and subjected tolimiting dilution to obtain monoclonal cell populations.

[0154] To select for cells permissive to EBO-Z virus entry, a separateculture of 6 batches of Jurkat-EctR cells (total of 1.2×10⁸ cells) weretransduced with HeLa cDNA library following the above protocol. After 2d, transduced cells were challenged with EBO-Z-blasti virus and selectedin blasticidin S. Viable cells were detected in selected cultures byTrypan blue exclusion, expanded, and grown by limiting dilution asmonoclonal cell populations.

[0155] Challenge of cells with pseudotype viruses andreplication-competent filoviruses. To determine permissivity to entry bypseudotype viruses, Jurkat-EctR cells were plated in 24-well dishes(200,000 cells/well), incubated with constant inocula of HIV-1 Luc⁺pseudotype viruses for 48 h, and luciferase expression was quantitatedas previously described ((Chan et al., (2000) J. Virol. 74, 4933-4937)).

[0156] To determine permissivity to entry by wildtype MBG virus,Vero-E6, parental Jurkat-EctR and reconstituted Jurkat-EctR (F10 clone)cells were inoculated with MBG (Musoke isolate) at increasingmultiplicity of infection (MOI) of 0.1, 1, and 10. On days 1,3 and 6post-infection cells were washed, dried on spot-slides, fixed withacetone, irradiated, and then immunostained with convalescent guinea pigantisera to MBG followed by FITC-conjugated anti-guinea pig antisera.Positive cells were counted by fluorescence microscopy.

[0157] To determine reconstitution of permissivity to MBG entry byexpression of FR-α, parental Jurkat-EctR cells (1.5×10⁷ cells) wereelectroporated (270 kV, 950 μF) with a mammalian expression vector(pCMV4neo) carrying no insert, a truncated (E4-1) cDNA, or full-lengthcDNA encoding FR-α. Transfection efficiency was quantitated in parallelusing an expression plasmid encoding GFP (pIRES2-EGFP) and assessingpercentage of GFP-positive cells by flow cytometry (10-20% positive).After recovery for 48 hours, transfected cells were plated in 24-wellplates (300,000 live cells/well), challenged with pseudotype luciferaseviruses, and luciferase expression was quantitated after 72 hours.

[0158] PCR recovery of retroviral library cDNA insert from transducedJurkat-EctR cells. To recover cDNA library inserts from Jurkat-EctR cellclones permissive to MBG entry, genomic DNA was extracted by the “EasyDNA” method as instructed by the manufacturer (Invitrogen). ExtractedDNA (50 ng) was used as template for PCR-based amplification using theExpand PCR kit (Roche Molecular Biochemicals, Indianapolis, Ind.) andoligonucleotide primers (Clontech, Palo Alto, Calif.) derived from theretroviral sequences flanking the cDNA inserts in the cDNA library.Specific DNA bands amplified in experimental samples, but not controlsamples, were extracted from agarose gels and used in conventional TAcloning steps using the pCR2.1 vector (Invitrogen). Insert sequenceswere verified by ABI Prism Dye terminator cycle sequencing(Perkin-Elmer, Foster City, Calif.) and were compared to known genomicand cDNA sequences using Entrez BLAST software.

[0159] To recover cDNA library inserts from Jurkat-EctR cell clonespermissive to EBO-Z entry, total RNA was extracted from cells by the RNASTAT 60 method (Tel-Test, Inc., Friendswood, Tex.). Using total RNA as atemplate, RT-PCR was performed using ALV reverse transcriptase kit asrecommended by the manufacturer (Roche Molecular Biochemicals) followedby PCR of resulting cDNA strands using the Expand PCR kit and primersderived from the same retroviral sequences flanking the library insertsas above. Subsequent cloning and sequencing of inserts were performed asdescribed previously.

[0160] Inhibition of pseudotype virus entry by blocking FR-α on targetcells. To cleave glycolipid membrane-anchored proteins such as FR-α fromthe cell surface, HeLa cells (30,000 cells/well in 24-well plates) orreconstituted Jurkat-EctR F10 cells (100,000 cells/well in 24-wellplates) were pre-incubated with phospholipase C (ICN Pharmaceuticals,Inc., Costa Mesa, Calif.) for 2 h at 37° C. Cells were washed withphosphate buffered saline (PBS) and challenged with pseudotypeluciferase viruses for 4 h at 37° C. Culture medium was then removed andreplaced, followed by assessment of luciferase expression after 72 h.

[0161] To block epitopes necessary for binding ligand to FR-α usingantibodies raised against FR-α, Jurkat-EctR F10 cells (100,000cells/well in 24-well plates) or Vero E6 cells (30,000 cells/well in24-well plates) were pre-incubated with media containing polyclonalrabbit anti-folate binding protein (FBP) or normal rabbit sera for 15minutes at 4° C. Cells were then challenged in the presence of antiserawith equivalent inocula of pseudotype luciferase viruses at 37° C.Luciferase expression was then quantitated after 72 h. Similarly,Jurkat-EctR F10 cells were pre-incubated with media containingmonoclonal mouse anti-FR (IgG1) or monoclonal mouse anti-HIV Gag p24(IgG1) for 15 minutes at 4° C. Cells were challenged with pseudotypeluciferase viruses in the presence of antisera and luciferase expressionwas assessed as above.

[0162] To determine if epitopes on FR-α which bind its natural ligandfolic acid are necessary for MBG or EBO-Z GP-mediated entry, viruschallenge of target cells was performed in the absence of fetal bovineserum which carries high concentrations of folic acid. Therefore, 12 hafter co-transfection of 293T cells with pNL-Luc E⁻R⁻ and envelope GPexpression vector to produce pseudotype luciferase viruses, media wasreplaced with RPMI 1640 containing no fetal bovine serum (FBS) and noexogenously added folic acid (Life Technologies, Inc., Grand Island,N.Y.), and virus supernatant was harvested after 36 h. HOS target cells(30,000 cells/well in 24-well plates) were pre-incubated with RPMI 1640in the absence of FBS and either in the absence or presence ofexogenously added folic acid (1 mg/L) for 15 minutes at 4° C. Cells werethen challenged with folic acid-free pseudotype luciferase viruses for 4h at 37° C. Virus was removed, replaced with RPMI 1640 containing 10%fetal bovine serum and exogenous folic acid (1 mg/L), and luciferaseexpression was quantitated after 72 h.

[0163] Inhibition of pseudotype virus entry by binding virion GP withsoluble FR-α (FBP). To competitively inhibit interaction ofvirion-anchored MBG or EBO-Z GP with membrane-anchored FR-α, solublebovine FBP (FBP, Sigma, 1 mg/ml reconstituted stock in PBS) waspre-incubated with pseudotype virus supernatant harvested in RPMI 1640in the absence of FBS or exogenous folic acid for 15 minutes at 4° C.HOS cells (30,000 cells/well in a 24-well plate) were challenged withvirus in the presence of FBP (10 μg/ml final concentration) for 4 h at37° C. Cells were then washed with PBS, media was replaced withDulbecco's Minimum Essential Medium containing 10% FBS, and luciferaseexpression was assessed after 72 hours.

[0164] Binding of membrane-anchored MBG GP with FBP. To provide furtherevidence that MBG GP binds FR-α, CHO-K1 cells, which do not expressdetectable levels of FR-α, were transiently transfected with MBG GP ornegative control Ampho GP (1 μg/well in a 6-well plate) usingLipofectAMINE as described by the manufacturer (Life Technologies,Inc.). After 12 hours, transfected cells were trypsinized and replated(70,000 cells/chamber) on 4-well Permanox chamber slides (Nalge NuncInternational, Naperville, Ill.) coated with poly-L-lysine (Sigma) aspreviously described (Allan, 2000) to ensure minimal cell detachmentduring staining. After 24 h, soluble bovine FBP (33 μg/ml) was incubatedin the presence of a polyclonal goat anti-bovine FBP (250 μg/ml), knownto be non-neutralizing for MBG entry, for 15 minutes at 4° C. in RPMI1640 containing no FBS or exogenous folic acid. Transfected CHO-K1 cellswere then incubated in the presence of the FBP/anti-FBP mixture or inthe presence of anti-FBP alone for 30 minutes at 4° C. Cells were washed3 times with ice cold PBS and fixed in 2% paraformaldehyde for 15minutes at 4° C. After 3 washes with ice cold PBS, cells were incubatedwith fluorescein-conjugated rabbit anti-goat IgG Fc secondary antibody(4 μg/ml)) for 45 minutes at 25° C. in the dark. Cells were washed 3times, mounted in Vectashield medium (Vector Laboratories, Inc.,Burlingame, Calif.), and analyzed for staining by fluorescencemicroscopy.

[0165] Results

[0166] A cDNA encoding the Marburg (MBG) or Ebola-Zaire (EBZ) envelopeglycoproteins (GP) was used to generate pseudotype viruses based on anenvelope-negative clone of the human immunodeficiency virus type 1(HIV-1) that had been engineered to contain a firefly luciferase gene(pNL4-3LucR-E-,provided by Dr. Nathaniel Landau, Salk Institute). Theenvelope cDNAs (pGEM-MBG, Xu et al., Nat. Med. 4(1):37-42, 1998;pGEM-EBO-Z, unpublished) were provided by Dr. Anthony Sanchez (SpecialPathogens Branch, CDC). By quantitating infections using this indicatorgene product, it was found that co-expressing the pNL4-3lucR-E andenvelope construct led to production of infectious virions with theproperties of the parental virus (Marburg or Ebola, respectively) (FIGS.1A-1C) Numerous human and other cell lines were then screened forpermissivity, and it was found that nearly all cells were permissivewith the exception of several human T-cell lines (including Jurkat,MT-2,SupT1 and C8166; FIGS. 2A and 2B) For subsequent cloningexperiments, a Jurkat derivative known as Jurkat-EctR (from Dr. GarryNolan, Stanford Univ) was used. Jurkat-EctR cells were modifiedgenetically by introduction of a cDNA library derived from HeLa cells.This library (purchased from Clontech, in the pLib Moloney-based vector)was introduced into Jurkat-EctR cells using a retroviral vector systemthat was optimized to achieve 30-40% transduction efficiency (FIG. 3).Cells that had been transduced with this library were “challenged” witha selectable MBG pseudotype virus. To prepare selectable pseudotypeviruses, a modified pseudotype strategy was used in which the MBG GP wasintroduced into 293T cells by co-transfection along with an alternateHIV-1 proviral clone pHIV-blasti (provided by Dr. Richard Sutton BaylorCollege of Medicine) containing the blasticidin S deaminase gene fromAspergillus terreus rather than the luciferase gene. Pseudotype virionsproduced from these co-transfections were then used to “challenge”Jurkat-EctR cells. Two days following challenge, the cells weretransferred into medium containing blasticidin S (ICN, 40 μg/ml) andmonitored for viability (FIG. 4). In negative control samples(pseudovirions lacking envelope GP), all cells died within 8 days and noviable cells were recoverable over a 3-week interval. In positivecontrol samples (pseudovirions containing envelope G-glycoprotein fromvesicular stomatitis virus,VSV), numerous viable cells were evident. Inthe experimental samples (pseudovirions containing MBG GP), smallnumbers of viable cells were detected. These cells were expanded foradditional study. In a key validation experiment, recovered cells werere-challenged with MBG GP pseudotype virus containing the luciferasegene, which confirmed that these cells were permissive for infection viathe MBG envelope, unlike the parental cells (FIG. 5). Subsequently,these cells were subjected to limiting dilution to obtain individualcell clones.

[0167] In the next step, individual MBG-permissive clones were used toisolate genomic DNA by the “Easy DNA” method (Invitrogen) method.Genomic DNA was used as template for PCR-based amplification usingoligonucleotide primers derived from the sequences flanking the cDNAinserts in the cDNA library. In experimental samples, but not controlsamples, specific DNA bands were amplified. These bands were extractedfrom agarose gels and used in conventional TA cloning steps using thepCR2.1 vector (Invitrogen). 12 Clones with independent inserts wererecovered, and automated DNA sequencing was used to determine thesequences of these inserts. Based upon sequencing from two directionsand assemblage of a contig based on overlapping sequences from thesesequences runs, one insert (clone 4-1) showed perfect identity with the3′ two-thirds of known cDNAs recognized as encoding human FR-α.

[0168] To verify that this molecule serves as a receptor for MBG, atransfection experiment was performed in which this cDNA (subcloned intoa mammalian expression vector, pCMV4Neo) was introduced transiently byelectroporation into Jurkat-EctR cells. These cells were then challengedwith pseudotype luciferase viruses and scored for luciferase signal as amarker of infection. Cells transfected with clone 4-1, but not emptyvector, exhibited a significant luciferase signal (FIG. 6). Finally, wefound that treatment of permissive cells with phospholipase C virtuallyabolished infection by MBG pseudotype, a finding that is consistent withthe GPI (glycophosphatidylinositol) type of membrane linkage that ischaracteristic of folate receptor-alpha (FIG. 7). These findings confirmthat the human FR-α as a receptor that mediates cellular entry by MBG.

[0169] Finally, pretreatments of JurkatEctR cells with a commerciallyavailable rabbit polyclonal antiserum raised against human folatebinding protein (Biogenesis), a cleaved form of full-length FR-α,completely abolished infection by MBG pseudotype virus. (FIG. 8). Toconfirm these findings in a naturally injectable cell type, monkey VeroE6 cells, which are typically used to passage MBG virus in cell culture(Peters et al., (1996) Filoviridae: Marburg and Ebola Viruses. FieldsVirology, Third Edition, eds. B N Fields, D M Knipe, P M Howley, et al.1161-1176) and express significant levels of FR-α by Northern blotanalysis, were challenged in the presence of anti-FBP. Entry by MBG wassubstantially inhibited by the anti-FBP antiserum (FIG. 9). Thus,similar inhibition of MBG entry was achieved on both geneticallyreconstituted human cells and untransduced monkey cells in the presenceof polyclonal anti-FBP, indicating that FR-α is important in infectionin different cell types and in different mammalian species.

[0170] To determine if other antibodies that recognize relevant epitopesof FR-α similarly abrogated MBG entry, Jurkat-EctR F10 cells werechallenged with pseudotype viruses in the presence of a monoclonalantibody preparation (No. 458) raised against human FR-α (FIG. 10). VSVentry was not inhibited in the presence of anti-FR-α compared withcontrol ascites fluid containing isotype-matched monoclonal antibodyrecognizing an irrelevant HIV p24 antigen, while MBG infection wasspecifically and potently reduced by anti-FR-α. The specific inhibitionof MBG entry by polyclonal or monoclonal antibodies raised separatelyagainst bovine FBP and human FR-α, respectively, further defines FR-α asa highly conserved mediator of MBG virus entry in multiple cell typesand mammalian species.

[0171] To test whether folic acid, a natural ligand of FR-α, is aspecific inhibitor of MBG entry, pseudotype luciferase viruses wereprepared in media containing neither fetal bovine serum (FBS) nor folicacid. Target cells (HOS) were then challenged with these viruspreparations in the presence or absence of folic acid and a reduced formof folic acid, 5-methyltetrahydrofolic acid (10 μM), which is known tobind at high affinity to FR-α Exposure to folic acid resulted in veryslight alteration of entry by negative control Ampho luciferasepseudotypes. However, MBG virus entry was reduced by nearly 80% in thepresence of folic acid (FIG. 11). This specific inhibition by folatecompounds indicates that FR-α is important in MBG virus entry.

[0172] Soluble FR-α inhibits MBG entry by interaction with virion GP.Another approach seeking to confirm a role for FR-α entailed usingsoluble FR-α to compete for the binding of MBG GP expressed on thevirion envelope. Secreted FBP purified from bovine milk waspre-incubated with pseudotype luciferase viruses prepared in medialacking FBS or folic acid. HOS cells were then inoculated with thesemixtures, and infection level was compared with that of uncomplexedvirus. The results are shown in FIG. 12. VSV entry was not significantlyaltered in the presence of FBP while MBG entry was inhibited by morethan 50% in the presence of FBP. Therefore, these results indicate thatFR-α is an important factor that binds MBG virions at the cell surface.

[0173] To obtain direct evidence that MBG GP can bind FR-α,immunofluorescence microscopy was used to visualize FBP bound to thesurface of cells expressing MBG GP. Since they express very low levelsof FR-α (Weitman et al., (1992) Cancer Res. 52, 3396-3401; Orr andKamen, (1995) Cancer Res. 55, 847-852), CHO-K1 cells were selected astarget cells and were transfected with expression vectors encodingeither MBG or Ampho GP. Two days following transfection, cells wereincubated with bovine FBP and an anti-bovine FBP antibody that we foundto be non-neutralizing for MBG entry. Samples were then fixed andstained with an anti-goat Ig fluorescein-conjugated secondary antibodyin order to highlight selectively those cells with FBP bound to theirsurface. Transfected cells did not stain significantly when exposed toanti-FBP and secondary antibody in the absence of FBP, indicating thatthe low endogenous levels of FR-α on CHO-K1 cells cause littlebackground staining. Additionally, cells transfected with Ampho GPexhibited only a low level of background staining in the presence ofFBP. In contrast, cells expressing MBG GP and incubated with FBPdisplayed a ring-like cell surface staining of significantly higherintensity. The significant and specific staining of cells expressing MBGGP by FBP provides direct evidence that MBG GP can bind FBP, whichfurther substantiates a role for FR-α in entry by MBG.

[0174] MBG GP and EBO-Z GP share common target cell factors formediating virus entry. We examined the mechanisms of target cell entryutilized by EBO. As with MBG, cellular entry controlled by the EBO-Z GPwas pH dependent (Wool-Lewis and Bates, (1998) J. Virol. 72, 3155-3160;Chan et al., (2000) J. Virol. 74, 4933-4937). Pre-treatment of HeLacells with phospholipase C abolished entry by EBO-Z pseudotype virus,but not by Ampho virus (FIG. 13A). These results raised the possibilitythat entry by EBO-Z is also mediated by a GPI-linked protein.

[0175] The genetic complementation protocol described earlier wasadapted for use in identifying host proteins that mediate entry by EBO-Zvirus. Previous studies had demonstrated that HeLa cells, but not Jurkatcells, are permissive to entry mediated by EBO-Z GP (Chan et al., (2000)J. Virol. 74 4933-4937). Therefore, after delivery of the retroviralHeLa cDNA library into Jurkat-EctR cells, transduced Jurkat-EctR cellswere challenged with pseudotype virus packaged by EBO-Z GP with thepHIV-blasti backbone (EBO-Z-blasti), and then selected in blasticidin S.Only the library-transduced cultures that had been challenged byEBO-Z-blasti virus yielded viable cells that survived selection.Subsequently, individual cell clones were expanded by limiting dilutionand re-challenged with pseudotype luciferase viruses. For example, cellclone A7-1, which had been selected for EBO-Z permissivity, was indeedinjectable by EBO-Z luciferase virus while parental cells were not (FIG.13B). Importantly, A7-1 was also found to be permissive for entry by MBGluciferase virus. Therefore, by genetically complementing the deficiencyof permissivity to EBO-Z virus entry, MBG entry was concurrentlyrestored. Similarly, the Jurkat-EctR F10 cell clone that had beenselected for MBG permissivity was injectable by MBG and EBO-Z luciferaseviruses (FIG. 13C). Thus, in two independent iterations of librarytransduction and virus challenge followed by selection, one approachutilizing MBG pseudotypes and the other utilizing EBO-Z pseudotypes,recovered cells were found to be permissive for both MBG and EBO-Zinfection. These results suggest that MBG and EBO-Z viruses depend on atleast one common factor in target cells to gain entry.

[0176] FR-α mediates entry by EBO-Z virus . To identify the cDNA insertin A7-1 cells responsible for reconstituting permissivity of MBG andEBO-Z infection, RT-PCR was performed using mRNA isolated from A7-1cells and primers recognizing the library retroviral sequences flankingthe insert. A cDNA insert carrying 100% identity with the full-lengthFR-α was isolated, including the natural methionine initiation codon,suggesting that FR-α mediates infection by EBO-Z as well as by MBGvirus.

[0177] To investigate the importance of FR-α for entry by EBO-Z virus,Jurkat-EctR F10 cells were challenged with pseudotype luciferase virusesin the presence of monoclonal anti-FR-α antibody or isotype-matchedcontrol antibody. While VSV control pseudotype infection levels wereunaffected, infection by EBO-Z pseudotype virus decreased significantlyin the presence of the anti-FR-α preparation (FIG. 13D). This resultprovides biochemical evidence that FR-α is a mediator of EBO-Z entry.

[0178] Further strategies to identify FR-α as a mediator of EBO-Zinfection included inoculating HOS cells with luciferase viruses in thepresence or absence of folic acid. While infection by Ampho virus didnot significantly decrease in the presence of folic acid, entry by EBO-Zvirus was inhibited significantly (FIG. 14A). In addition, viruschallenge of HOS cells in the presence of FBP caused no significantdecrease in entry by VSV pseudotypes but a substantial inhibition ofEBO-Z infection (FIG. 14B). Therefore, mirroring the inhibition profilesobserved for MBG virus, EBO-Z pseudotype virus entry was specificallyabrogated in the presence of agents expected to disrupt interactionsbetween virion GP and FR-α.

[0179] Finally, to assess the role of FR-α in mediating EBO-Z entry innaturally permissive cell types routinely used for study of filovirusinfection (Peters et al., (1996) Filoviridae: Marburg and Ebola Viruses.Fields Virology, Third Edition, eds. B N Fields, D M Knipe, P M Howley,et al. 1161-1176.), Vero E6 cells were challenged with EBO-Z luciferasevirus in the presence of either polyclonal rabbit anti-bovine FBPantibody or normal rabbit sera. VSV entry decreased marginally in thepresence of anti-FR-α, while EBO-Z entry was completely abrogated in thepresence of the anti-FR-α preparation (FIG. 14C). Therefore, as with MBGinfection, EBO-Z entry was specifically inhibited in the presence ofmonoclonal or polyclonal anti-FR-α antibodies, folic acid, or solubleFBP. Considered together with the recovery of independent cDNA insertsencoding FR-α from separate library transductions challenged with eitherMBG or EBO-Z-blasti viruses, we conclude that FR-α is a cofactor forcellular entry by either MBG or EBO-Z virus.

[0180] The above results and discussion indicate that the subjectinvention provides important new means of modulating and even inhibitingfilovirus entry into permissive cells. As such, the subject inventionprovides new means of treating the devastating illness mediated byfiloviruses. Therefore, the subject invention represents a significantcontribution to the art.

[0181] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0182] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A method of at least slowing the progression of afilovirus associated disease condition in a host, said methodcomprising: administering to said host an effective amount of an agentthat at least reduces the amount of folate receptor mediated filoviruscell entry in said host; whereby the progression of said filovirusassociated disease condition is at least slowed.
 2. The method accordingto claim 1, wherein said agent is a folate receptor antagonist.
 3. Themethod according to claim 1, wherein said agent is a filovirus-derivedfolate receptor binding fragment, derivative or mimetic thereof.
 4. Themethod according to claim 1, wherein said agent is a soluble folatereceptor.
 5. The method according to claim 1, wherein said agentmodifies expression or configuration of membrane bound folate receptorsso as to at least reduce their filovirus binding activity.
 6. The methodaccording to claim 1, wherein said agent is a monoclonal antibody,polyclonal antibody, mixture of antibodies, or binding fragment ormimetic thereof.
 7. The method according to claim 1, wherein said agentat least reduces the expression of membrane bound folate receptors insaid host.
 8. The method according to claim 1, wherein said agent is anagent that modulates trafficking, clustering or internalization ofmembrane bound folate receptors in said host.
 9. The method according toclaim 1, wherein said host is a mammal.
 10. The method according toclaim 9, wherein said mammal is a human.
 11. The method according toclaim 1, wherein said method treats a filovirus associated diseasecondition.
 12. The method according to claim 1, wherein said methodprevents a filovirus associated disease condition in a mammalian host.13. A method for determining the ability of a test compound to at leastslow the progression of a filovirus associated disease condition, saidmethod comprising: contacting a folate receptor or filovirus bindingderivative or mimetic thereof with a filovirus or folate receptorbinding derivative or mimetic thereof and said test compound; anddetermining the effect of said compound on the binding interactionbetween said folate receptor and said filovirus; whereby the ability ofsaid test compound to at least slow the progression of a filovirusassociated disease condition is determined.
 14. The method according toclaim 13, wherein said method is a cell based method, and said folatereceptor is membrane bound and presented on the cell surface.
 15. Themethod according to claim 13, wherein said method is a cell free method.16. A pharmaceutical preparation for use in at least slowing theprogression of a filovirus mediated disease condition, said preparationcomprising: an effective amount of an agent that at least reduces theamount of folate receptor mediated filovirus cell entry in a mammalianhost.
 17. The pharmaceutical preparation according to claim 16, whereinsaid agent is a folate receptor antagonist.
 18. The pharmaceuticalpreparation according to claim 16, wherein said agent is a solublefolate receptor or a filovirus binding fragment, derivative or mimeticthereof.
 19. The pharmaceutical preparation according to claim 16,wherein said agent modifies expression or configuration of membranebound folate receptors so as to at least reduce their filovirus bindingactivity.
 20. The pharmaceutical preparation according to claim 16,wherein said agent is a monoclonal antibody, polyclonal antibody,mixture of antibodies, or binding fragment or mimetic thereof.
 21. Thepharmaceutical preparation according to claim 16, wherein said agent atleast reduces the expression of membrane bound folate receptors in saidhost.
 22. The pharmaceutical agent according to claim 16, wherein saidagent is an agent that modulates trafficking, clustering orinternalization of membrane bound folate receptors in said host.
 23. Amethod for immunizing a host against a filovirus mediated diseasecondition, said method comprising: administering to said host aneffective amount of an immunogen that causes said host to mount animmune response against membrane bound folate receptors.
 24. The methodaccording to claim 23, wherein said immunogen is a membrane bound folatereceptor or fragment thereof.
 25. The method according to claim 23,wherein said immunization is passive immunization.
 26. A method of aidentifying a cell surface receptor used by a virus for entry into acell, said method comprising: (a) identifying a cell line that isnon-permissive for entry of said virus; (b) transfecting a population ofsaid non-permissive cell line with a genomic library obtained from acell line permissive for entry of said virus; (c) identifying at leastone cell from said transfected cell population which is permissive forentry of said virus; and (d) identifying at least one gene of saidpermissive cell line in the genome of said transfected permissive cell.